Some of the hepatocellular carcinoma lines in which we had studied the effect of deferoxamine (desferrioxamine; DFX) contained integrated hepatitis B virus, which has an unusual reverse transcription step in its replication; this promoted us to test DFX for antiretroviral activity. Duplicate cultures of H9 cells infected with HIV-1 were studied. At day 7, coded samples of supernatants were tested. In this blinded study, DFX inhibited the expression of p24 antigen and substantially reduced detectable levels of gag and env genes in H9 cell cultures after 7 days. The inhibition was dose-dependent; 30 mu-m DFX had the same effect on p24 expression as 187 mu-M AZT (azidothyimidine [AZT]; zidovudine) (50 mu-g/ml). Cultures grown in medium lacking DFX and AZT produced substantial concentrations of p24, and the signals for gag and env sequences were strongly positive. Viability of the H9 cells was above 70% at day 7 in all cultures. Three independent experiments were done, with similar results for p24 expression. Evaluation of gag and env were available only in one experiment. DFX could have inhibited HIV-1 by interfering with the RNA-dependent DNA synthesis that occurs early in each infectious cycle. The observation of in vitro inhibition of HIV-1 by DFX suggests a new mechanism of viral inhibition.